Enferm Infecc Microbiol Clin.

2017;35(6):367–376

www.elsevier.es/eimc

Continuing medical education: Methods of rapid diagnosis

Rapid diagnosis of gastrointestinal tract infections due to parasites,

viruses, and bacteria夽
Luz Balsalobre-Arenas a , Teresa Alarcón-Cavero a,b,∗
a
Microbiology Department, Hospital Universitario de La Princesa, Sanitaria Princesa Research Institute, Madrid, Spain
b
Department of Preventive Medicine, Public Health and Microbiology, School of Medicine, Autonomous University of Madrid, Madrid, Spain

a r t i c l e i n f o a b s t r a c t

Article history: Rapid diagnostic techniques are valuable tools in the diagnosis of gastrointestinal infections, especially
Received 11 January 2017 for the detection of some microorganisms and in certain groups of patients. While antigen detection
Accepted 14 January 2017 techniques are widely used in Clinical Microbiology laboratories, for the diagnosis of viruses, some
Available online 19 May 2017
parasites and some bacteria, molecular techniques are routinely used only for some pathogens (such
as Clostridium difficile). However, molecular techniques are constantly evolving, and they allow a rapid
Keywords: diagnosis for an increasing number of pathogens, with high sensitivity and specificity. In addition, they
Rotavirus
are also able to detect virulence factors or resistance mechanisms. Syndromic surveillance systems,
Clostridium difficile
Helicobacter pylori
which detect different pathogens simultaneously, are very promising because they enable the most
frequent pathogens to be diagnosed in a few hours and they can be very useful in certain patients.
For the diagnosis of Helicobacter pylori infection, molecular techniques are able to detect bacteria and
its resistance to clarithromycin and levofloxacin, allowing the most appropriate treatment to be selected
for each patient when bacterial culture is not possible.
© 2017 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiologı́a
Clı́nica. All rights reserved.

Diagnóstico rápido de las infecciones del tracto gastrointestinal por parásitos,

virus y bacterias

r e s u m e n

Palabras clave: Las técnicas de diagnóstico rápido son herramientas de gran valor en el diagnóstico de las infecciones gas-
Rotavirus trointestinales, especialmente para la detección de algunos microorganismos y en determinados grupos
Clostridium difficile de pacientes. Mientras que las técnicas de detección de antígeno son práctica habitual en los laboratorios
Helicobacter pylori
de microbiología clínica para el diagnóstico de virus, algunos parásitos y algunas bacterias, las técnicas
moleculares se utilizan de manera rutinaria solo para determinados patógenos (como Clostridium difficile).
Sin embargo, son técnicas en constante evolución que permiten el diagnóstico rápido de un número cada
vez mayor de patógenos con una elevada sensibilidad y especificidad y también permiten la detección de
factores de virulencia o mecanismos de resistencia. Los sistemas de diagnóstico sindrómico, que detectan
diferentes patógenos de forma simultánea, son muy prometedores porque permiten diagnosticar en pocas
horas los patógenos más frecuentes y pueden ser de gran utilidad en determinados pacientes.

DOI of original article: http://dx.doi.org/10.1016/j.eimc.2017.01.002

夽 Please cite this article as: Balsalobre-Arenas L, Alarcón-Cavero T. Diagnóstico rápido de las infecciones del tracto gastrointestinal por parásitos, virus y bacterias. Enferm
Infecc Microbiol Clin. 2017;35:367–376.
∗ Corresponding author.
E-mail address: talarcon@helicobacterspain.com (T. Alarcón-Cavero).

2529-993X/© 2017 Elsevier España, S.L.U. and Sociedad Española de Enfermedades Infecciosas y Microbiologı́a Clı́nica. All rights reserved.
368 L. Balsalobre-Arenas, T. Alarcón-Cavero / Enferm Infecc Microbiol Clin. 2017;35(6):367–376
Para el diagnóstico de la infección por Helicobacter pylori las técnicas moleculares, que pueden detectar
tanto la bacteria como su resistencia a claritromicina y levofloxacino, permiten seleccionar el tratamiento
más adecuado para cada paciente cuando el cultivo de la bacteria no es posible.
© 2017 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiologı́a Clı́nica.
Introduction
Infections of the gastrointestinal tract include 2 distinct clin-
ical entities: gastric infections caused by Helicobacter pylori, and
gastroenteritis, inflammation of the gastric and intestinal mucosa,
which may be caused by various pathogens (bacteria, viruses, or
parasites) or their toxins, and which includes diarrhoea, vomi-
ting, abdominal pain and/or fever. Different pathogens may give
rise to similar symptoms, making the aetiological diagnosis impor-
tant. They are often self-limiting processes, but may present high
morbidity and mortality rates, especially in young children or
immunosuppressed patients.1 In developing countries they are
associated with poor sanitation, lack of running water and nutri-
tional deficiencies in the population, and present higher morbidity
and mortality rates than in developed countries, where they are
frequently associated with food-borne infections.2,3
The rapid and successful diagnosis of the pathogen involved
in the clinical picture is important since some pathogens require
a specific treatment; with some it is necessary to use control
measures to avoid dissemination, and in some cases knowing the
pathogen will enable a decision to be taken as to whether to hos-
pitalise the patient or treat them on an outpatient basis.1,4
Types of rapid techniques
Conventional diagnostic methods used in the diagnosis of gas-
troenteritis include bacterial culture techniques, which require
several days to obtain results, microscopic tests for detection of
eggs and parasites, requiring high specialisation of personnel, and
antigen detection techniques.1 Frequently, when the aetiologic
agent causing the clinical picture is successfully cultivated, the
symptoms have already been resolved and it is not useful for the
management of the patient.
Rapid techniques based on microscopic observation
Gram staining, a technique used in most clinical specimens, is
not useful in faecal samples, as they will stain the usual microbiota
bacteria. It does allow for observation of the characteristic curved
structures of Campylobacter, which may have value in certain cir-
cumstances, although it is not performed routinely.2 Microscopic
examination for leukocytes is non-specific, but may be helpful if
invasive enteritis is suspected (Table 1).
Rapid techniques based on antigen-antibody reaction
The microbial antigens present in the biological samples can
be detected and quantified by immunological techniques based on
the specificity of the antigen-antibody reactions. A wide variety of
immunoassays are available for the diagnosis of gastrointestinal
tract infections. Some, such as immunofluorescence or enzyme-
linked immunosorbent assay (ELISA), are laborious and require
trained personnel, while others, such as membrane EIAs, lateral
Todos los derechos reservados.
flow immunoassays or immunochromatographic tests (LFIA/ICT),
and agglutination techniques, are extremely simple and fast.
ELISA enzyme-linked immunosorbant assay
They use specific antibodies for the test antigen, generally
bonded to the wells of a microplate. The antigen present in the clin-
ical sample is combined with the antibody bound to the solid phase
and its presence is detected by the addition of a second antibody
conjugated to an enzyme. The most frequently used enzymes are
peroxidase and alkaline phosphatase. When the enzyme acts on the
substrate, a colorimetric signal will be produced that is visible to the
naked eye or quantifiable through the use of a spectrophotometer.
In general, the sensitivity and specificity values of these techniques
are excellent. They allow simultaneous processing of batches of
samples and, in some cases, a quantification of the detected anti-
gen. They are laborious techniques as they require the addition of
multiple reagents, wash steps and incubation times. However, they
are easily automated.
Membrane enzyme immunoassay
The capture antibody is immobilised in a membrane through
which the sample flows. If the antigen is present, it will be retained
by said antibody, and the antigen-antibody binding will become
apparent, as in a conventional ELISA, through a second antibody
conjugated to an enzyme. The interaction of the enzyme and its
substrate causes a colour change. A determination is considered
positive when coloured bands are obtained both in the control line
(where the conjugate is attached) and in the reaction line (where
the antigen is attached). Through this type of assay, the samples
are processed individually, no equipment or specialised personnel
are required. The results are obtained approximately in 15–20 min,
and are easily interpreted.
Immunochromatographic tests or lateral flow immunoassay
They consist of nitrocellulose or nylon membranes, like a strip
or cassette, through which the sample flows by capillary action. In
the most common sandwich assays, two zones are distinguished on
the strip: the reaction zone, where antibodies against the test anti-
gen are immobilised, and the control zone, where anti-conjugated
antibodies are immobilised. The conjugate is an antibody, specific
to the test antigen, labelled with a colloidal gold molecule or with
coloured polystyrene microspheres. If the antigen is present in the
sample, it will bind to both the conjugated antibody and the capture
antibody immobilised in the reaction zone. The excess conjugated
antibody will continue to migrate through the membrane until it is
retained in the control zone. In negative samples, only the control
zone will be coloured, whereas if the sample is positive, both the
control and reaction zones will be coloured.5 These are qualitative
techniques, very fast and simple to perform, which do not require
special laboratory equipment and the samples are processed indi-
vidually. Most present high values of sensitivity and specificity,
although generally lower than those presented by conventional
ELISA techniques.
 L. Balsalobre-Arenas, T. Alarcón-Cavero / Enferm Infecc Microbiol Clin. 2017;35(6):367–376
Table 1
Types of diarrhoea in terms of mechanism of action.
Gastroenteritis Symptoms Location
Secretor Liquid Proximal small
diarrhoea intestine
Inflammatory/invasive Dysentery Colon
Latex agglutination techniques
They are based on the use of latex particles bound to the crys-
tallisable fragment (Fc) of immunoglobulins. The antigen-binding
fragments (Fab) are exposed and capable of binding to the anti-
gen found in the sample. When the antibodies bind to the antigen,
a lattice of the latex particles is produced which results in visi-
ble agglutination. These types of techniques require the specimens
to be processed individually, are easy to perform, fast and do not
require special equipment. However, cross reactions may occur
with other sample antigens or prozone phenomena, in which an
excess of antibodies prevents the formation of the framework.
Direct immunofluorescence
It is based on the detection of antigens by the use of
fluorochrome-labelled antibodies on extensions of clinical sam-
ples prepared on a slide. The extension is fixed, incubated with the
antibody specific to the fluorochrome-conjugated test antigen and,
after washing, the results are read by fluorescence microscopy. Its
main disadvantages are the cost of the reagents and the laborious-
ness of the technique.
Rapid molecular techniques
Molecular techniques have revolutionised microbiological diag-
nosis and, despite their high cost, represent an interesting
alternative to conventional methods due to their speed and high
sensitivity and specificity. Polymerase chain reaction (PCR) repro-
duces the physiological process of DNA duplication in cells in vitro,
exponentially amplifying a specific sequence of double-stranded
DNA.
Since its invention, different variants of PCR have been designed
that improved its diagnostic yield.
PCR after reverse transcription or RT-PCR is used for the detec-
tion and amplification of RNA. The RNA present in the sample
is used as a template for synthesising the complementary DNA
(cDNA) by inverse transcriptase or reverse transcriptase, and the
resulting DNA is amplified by standard PCR.
Quantitative PCR (qPCR) or real-time PCR is much faster than
the conventional version, and provides a continuous and precise
quantification of the DNA that is being formed.6 In order to quantify
the DNA or RNA present in the sample, it is necessary to perform a
standard parallel curve.
Multiplex PCRs enable, through the use of multiple probes or
pairs of primers, the simultaneous detection of different pathogens.
These systems allow a syndromic diagnosis of gastrointestinal dis-
ease by simultaneously detecting the most frequently involved
enteropathogens, including bacteria, viruses and parasites.
Other amplification techniques are available, such as isothermal
amplification, in which nucleic acid amplification takes place at a
369
Characteristics Bacteria
No leukocytes Vibrio cholerae
Action of toxins ETEC
Staphylococcus aureus
Bacillus cereus
Clostridium perfringens
Leukocytes Shigella
Invasion or cytotoxin STEC
(causes damage to the Salmonella (non-typhoidal)
mucosa and causes Vibrio parahaemolyticus
inflammation) Clostridium difficile
Campylobacter
constant temperature with the involvement of different enzymes
such as reverse transcriptase, RNAse H, helicase or RNA polymerase.
Rapid techniques for the detection of individual pathogens
Diagnosis of parasites
Traditionally, the microbiological diagnosis of both protozoa
(amoebas, flagellates, ciliates, coccidia and microsporidia) and
helminths (nematodes, cestodes and trematodes) has been per-
formed by microscopic identification of the parasitic forms that are
expelled in the patient’s faeces. To increase the diagnostic yield, it
is necessary to use concentration techniques and, in some cases,
specific stains. These determinations only permit the diagnosis of
the acute and patent infection, require the taking of serial sam-
ples, are very laborious, require specialised personnel and present
limitations in terms of sensitivity and specificity. Screening tests
for parasitic antigens in faeces use specific antibodies that recog-
nise secretion, surface, or wall molecules of parasites.7 There are
commercialised systems that allow for the simultaneous detec-
tion of 2 or 3 different species (Table 2). The amplification tests of
genomic sequences are those of greater sensitivity and specificity,
and thanks to the constant characterisation of genomes of differ-
ent organisms, it is possible to design PCR protocols for each of the
parasites of interest. However, by using these techniques only one
or a few parasites can be detected at a time and there are very few
marketed.8
Entamoeba histolytica
Entamoeba histolytica causes amoebic dysentery and is one of
the possible causes of traveller’s diarrhoea. The traditional diagno-
sis of amoebiasis has been based for many years on microscopy.
However, microscopic techniques do not differentiate E. histolytica
from the commensal amoebae Entamoeba dispar and Entamoeba
moshkovskii. PCR techniques, based mostly on the amplifica-
tion of coding regions of the small 18S ribosomal subunit (18S
rRNA), differentiate the 3 species of morphologically indistinguish-
able Entamoeba and present excellent levels of sensitivity and
specificity.9 On the other hand, the detection of E. histolytica anti-
gens by ELISA is a good option if molecular methods are not
available. Currently, different membrane EIAs and marketed ICT
assays are available that detect E. histolytica-specific antigens in
faeces and in abscess content.10,11 These assays are based on the
detection of specific epitopes of amoebic lectin and have a sensi-
tivity higher than that of microscopic examination.10,12 Some of the
marketed assays only allow recent or frozen stools to be used.
370 L. Balsalobre-Arenas, T. Alarcón-Cavero / Enferm Infecc Microbiol Clin. 2017;35(6):367–376

Table 2 Helminths
Commercialised assays, based on the detection of coproantigens, for the diagnosis
There are 3 groups of helminths of medical importance: nema-
of gastrointestinal tract infections caused by protozoa.
todes (roundworms), cestodes (bandworms) and trematodes (or
Protozoa Diagnosis method Manufacturer staves). The diagnosis of some cestodes is based on the obser-
Entamoeba histolytica ELISA Cellabs vation of proglottids or segments. However, in most helminth
Remel infections, diagnosis is made by microscopic identification of their
TechLab eggs or larvae in the patient’s stool. Although there are also special
Membrane EIA TechLab
techniques, valid only for certain helminths, such as the Graham
ICT CerTest biotec
Giardia lamblia ELISA Cellabs ribbon in the diagnosis of Enterobius vermicularis or the cultiva-
Remel tion of Strongyloides stercoralis, advances in the rapid diagnosis
TechLab of intestinal helminths are not as significant as in protozoa. Anti-
ICT CerTest biotec
gen detection tests have not been highly developed and, although
Remel
IFD Cellabs several studies have been published,14 they have not yet been
Cryptosporidium ELISA Remel marketed. More frequent are the serological tests of specific anti-
TechLab bodies, available for the diagnosis of hydatidosis, cysticercosis,
ICT CerTest biotec fasciolosis, schistosomiasis, toxocariasis, anisakiasis, filariasis and
Remel
strongyloidiasis. Some of these assays have been marketed, while
IFD Cellabs
G. lamblia and ELISA Remel others are only carried out at reference centres. They all have the
Cryptosporidium Membrane EIA TechLab drawback of not distinguishing between current and past infection,
ICT Becton so they are more useful in non-endemic areas, and often have cross-
Dickinson
reactivity problems15,16 As for the molecular diagnosis, a multitude
CerTest biotec
Meridian
of PCR protocols have been designed, both conventional and real-
Bioscience time, for the diagnosis of helminthiasis.8 However, so far none has
Remel been marketed.
IFD Cellabs
Medical
Diagnosis of virus
Chemical
Corporation
Meridian The main viruses producing gastroenteritis in humans are
Bioscience rotavirus, astrovirus, adenovirus and norovirus. Others such
Cryptosporidium, G. ICT CerTest biotec
as coronaviruses, torovirus, picobirnaviruses and picornaviruses
lamblia and E. TechLab
histolytica/dispar
(Aichi virus) can also cause gastrointestinal tract infection, but with
a much lower frequency.16 The rapid diagnosis of viral gastroenteri-
tis is made through the detection of viral antigens (Table 3) or by
molecular methods based on the detection of specific genes.
Giardia lamblia
Giardiasis, caused by Giardia lamblia, is a parasitic disease of Rotavirus
great epidemiological and clinical importance due to its high Rotaviruses are highly contagious RNA viruses that are trans-
prevalence and pathogenicity, mainly among children. Various mitted by the faecal-oral route, by fomites or by the respiratory
methods of antigen detection with sensitivity and specificity levels route. According to the antigenic characteristics of the VP6 glyco-
superior to those of microscopic examination have been mar- protein, a component of the internal virion capsid, 8 serogroups
keted for diagnosis. These tests are also useful in screening the (A-H) are distinguished. Human infections are mainly produced by
infant population and as a treatment control to confirm healing. rotavirus serogroup A and, to a lesser extent, by serogroups B and
The various EIAs marketed use monoclonal or polyclonal anti- C. The VP4 protein, which constitutes the virion spicules, and the
bodies to detect G. lamblia-membrane antigens such as GSA 65 VP7 glycoprotein, constituent of the outer capsid, determine the
or CWP1.11 They are rapid techniques, which are cost-effective existence of the serotypes P and G, respectively.17 The diagnosis of
and allow for the processing of recent stools or faeces preserved serogroup A rotavirus infections is based on the detection of viral
in formalin. For G. lamblia commercially available ICT assays antigens in stool samples, specifically the VP6 glycoprotein. At the
are also available. However, most require using fresh or frozen moment there are no commercially available methods to detect
stools, without preservatives, and have a lower sensitivity than rotavirus from other serogroups. The formats available are highly
ELISA assays. Direct immunofluorescence (DFI) stains for G. lam- varied: ELISA, membrane EIA, ICT and latex agglutination.18–20 The
blia using a GSA 65 antigen-specific monoclonal antibody are also membrane EIA, the ICT techniques and latex agglutination are suit-
available. able methods for hospitals with few or sporadic samples. However,
they are less sensitive than conventional ELISAs. The more sensi-
tive and specific molecular methods are based on an RT-PCR. For
Coccidia the diagnosis of group A rotavirus by RT-PCR, marketed systems
Cryptosporidium hominis and Cryptosporidium parvum are the 2 use specific primers of the gene encoding the NSP3 non-structural
main pathogenic species of human parasite. Fundamentally they protein. This methodology also allows the detection of rotavirus of
affect immunosuppressed patients and cause severe chronic diar- different serogroups.21
rhoea. Coccidiosis is traditionally detected through the microscopic
demonstration of oocysts following special stains such as Kinyoun Astrovirus
or auramine-rhodamine. There are various other antigen detec- Astroviruses are non-enveloped viruses with a positive-sense
tion techniques that are valid alternatives, such as IFD, ELISA or single-stranded RNA genome, small (28–41 nm in diameter) and
ICT techniques, that have shown specificity and sensitivity values star-shaped under an electron microscope. There are 8 differ-
higher than those of microscopic observation.7,11,13 However, none ent serotypes (1–8) but, due to the existence of antigenic cross
of the methods mentioned allows the identification of species, so reactions, it is possible to detect all of them using a group-
it is necessary to resort to PCR techniques. specific antibody called MAb8E7, capable of recognising an epitope
 L. Balsalobre-Arenas, T. Alarcón-Cavero / Enferm Infecc Microbiol Clin. 2017;35(6):367–376 371

Table 3 are very useful in the study of epidemic outbreaks, but not so much
Commercialised assays, based on the detection of coproantigens, for the diagnosis
in the diagnosis of sporadic cases. However, the available ICTs and
of gastrointestinal tract infections caused by viruses.
membrane EIAs have high sensitivity and specificity values.
Virus Diagnosis method Manufacturer

Rotavirus ELISA Bio-Rad Diagnosis of bacteria

Meridian
Bioscience Toxigenic Clostridium difficile
r-biopharm
Clostridium difficile is a spore-producing anaerobic gram-
ICT CerTest biotec
Coris positive bacillus found in the soil and the intestinal tracts of
bioConcept animals and humans and can colonise up to 50% of children under
Meridian one year old, 3–5% of healthy adults, and a higher percentage
Bioscience
of hospitalised adult patients or those in nursing homes. C. diffi-
r-biopharm
Thermo cile produces antibiotic-associated diarrhoea, but symptoms may
Scientific range from asymptomatic carriers, to moderate or mild diarrhoea
Latex agglutination Bio-Rad and pseudomembranous colitis.2,24,25 Pathogenic strains have a
Astrovirus ELISA r-biopharm pathogenicity locus that codes for toxin A, an enterotoxin encoded
ICT CerTest biotec
by the TcdA gene, and toxin B, a potent cytotoxin encoded by the
Adenovirus ELISA Meridian
Bioscience TcdB2,25 gene, although they may not present toxin A and only toxin
r-biopharm B. The hypervirulent strain NAP1/027/B1 has been associated with
ICT CerTest biotec increased morbidity and mortality rates and produces greater toxin
Norovirus genotypes I ELISA r-biopharm
expression, more efficient sporulation and expression of a binary
and II ICT CerTest biotec
r-biopharm
toxin, although not all the strains currently detected have these
Rotavirus and ICT bioMérieux virulence characteristics.2,26
Adenovirus CerTest biotec In patients with risk factors for C. difficile diarrhoea, both hos-
Coris pitalised and non-hospitalised, rapid detection of toxins A and B
bioConcept
should be performed to initiate antibiotic treatment and to apply
r-biopharm
Rotavirus and ICT CerTest biotec control measures to prevent it spreading to other patients.
Norovirus The most commonly used methods are:
Rotavirus, Adenovirus, ICT CerTest biotec Antigen detection. Detection of glutamate dehydrogenase (GDH)
Astrovirus and
by EIA or latex particles. GDH is a very abundant antigen of the
Norovirus
cell wall of C. difficile and its detection is very sensitive but not
very specific, since it is present in both toxigenic and non-toxigenic
strains.2,24,27
Detection of toxins A and/or B. It can be done by cytotoxicity tests
or by ICT or EIA techniques.4,28 The cytotoxicity test is considered
common to all human serotypes. Several commercial ELISA
the reference method for detection of toxin B in the faeces, with
kits are currently available to allow the detection of human
a high sensitivity and specificity, but it is very laborious, in addi-
astrovirus in faeces. The diagnosis can also be established by
tion to not being a rapid test. There are numerous commercial EIA
RT-PCR.22
tests for the detection of toxins A, B or both. It is recommended
to use EIA techniques that detect toxin B or both, because of the
Adenovirus producing strains of toxin B. However, these commercial tech-
Enteric adenoviruses of serotypes 40 and 41 are the most niques have low sensitivity compared to cell cultures, so nucleic
frequently associated with childhood gastroenteritis. There are acid detection techniques must be used.2,28 The detection of toxin
specific type methods that detect only serotypes 40 and 41 and from the isolation obtained by culture should not be used as a sin-
other specific group methods that detect all adenoviruses. The gle method, since it requires several days to grow, but its use, in
diagnosis of enteric adenovirus infections is usually established addition to the direct detection of toxin, can increase the positivity
by ELISA, latex agglutination or ICT. Some of these techniques by 10%.29
allow simultaneous detection of adenovirus and rotavirus. There Detection of toxin A/B genes by nucleic acid amplification. It is the
are also commercially available highly sensitive and specific PCR technique that offers greatest sensitivity and specificity, although
methods that allow the detection and characterisation of enteric it is more expensive. There are different real-time PCRs that allow
adenoviruses.16 detection of C. difficile, toxin B, and binary toxin in 90 min. GeneX-
pert presents a 98% level of accordance with the reference test and
Norovirus greater sensitivity than the ICT.4,30 However, a sample can be posi-
Noroviruses are the leading cause of non-bacterial gastroenteri- tive by molecular method and negative by EIA and cell culture, and
tis in individuals of all ages and are the most common cause of as a result doubt can arise from the clinical meaning.2,27
outbreaks of acute gastroenteritis. They are non-enveloped viruses, To arrive at a correct diagnosis and to be cost-effective, one of
with a diameter of approximately 38 nm, whose genome is made the following algorithms must be used2,24,25 : (1) perform a GDH
up of positive-sense single-stranded RNA. Of the 5 genogroups EIA and study the presence of toxins using the molecular method
described, only genogroups I, II and IV have strains that infect when positive, (2) perform a combination of GDH and toxin A/B
humans. RT-PCR has become the reference technique for its diag- EIA simultaneously and confirm the samples showing discrepant
nosis. The primers employed enable amplification of the viral RNA results using the molecular method or (3) perform only the molec-
polymerase gene or the gene encoding the capsid proteins. Real- ular method on all samples.
time RT-PCR is more sensitive and allows to detect and differentiate
between genogroups I and II more quickly. In addition, there are Escherichia coli
several diagnostic tests based on the detection of antigens.23 Some Escherichia coli may be part of the normal intestinal microbiota,
ELISAs have a high specificity but a moderate sensitivity, so they but some variants (considered pathovarieties or pathotypes) have
372 L. Balsalobre-Arenas, T. Alarcón-Cavero / Enferm Infecc Microbiol Clin. 2017;35(6):367–376
the capacity to produce diarrhoea and are a cause of morbidity and
mortality around the world.31 They are defined by the expression of
one or more virulence factors: enteropathogen (EPEC), Shiga toxin
producer (STEC), also called enterohemorrhagic or verocytotoxin
producer, which includes serotype O157, enteroinvasive (EIEC),
enterotoxigenic (ETEC) enteroaggregative (EAEC) and adherent-
invasive (AIEC).2,31
In most clinical and public health laboratories only STEC is stud-
ied and is defined by the presence of Shiga toxin 1 (stx1) and/or
Shiga toxin 2 (stx2) genes. The culture allows only E. coli O157 to be
detected, but culture-independent methods detect both O157 and
the others.2 The CDC recommends simultaneously preparing the
differential culture to detect E. coli O157 and the molecular study
for the detection of stx1 and stx2 in faeces of patients with acute
diarrhoea acquired in the community and with possible haemolytic
uraemic syndrome.32 If a molecular method is not available, it
should be sent to a reference centre.
The following can be used: (1) EIA Stx, which can detect and in
some cases differentiate between stx1 and stx2, from an enriched
broth incubated overnight at 37 ◦ C, of both STEC O157 and non-
O157, and (2) molecular techniques, which detect the stx1 and
stx2 genes directly from faeces, and which are the most sensitive
method.2 Recently the use of a new ICT performed directly on stool
samples with excellent results has been published.30
Campylobacter
Campylobacter antigen detection tests have been developed
from stool samples, in ELISA format, on a microwell plate or in lat-
eral flow ICT format. Although they are faster methods than the
culture, they are less sensitive and do not differentiate between
Campylobacter jejuni and Campylobacter coli. There are molecular
methods that can be performed directly from faeces, which are the
most sensitive, but are not sufficiently validated.2
Rapid techniques for detection of other diarrhoea-producing
bacteria
A rapid test (ICT, dipstick) detects Shigella from colonies, and
directly from faeces or rectal exudates, thanks to monoclonal
antibodies against lipopolysaccharide.4 The enterotoxins of Staphy-
lococcus aureus, Bacillus cereus and Clostridium perfringens can be
detected in food, faeces or in strains cultured by commercial meth-
ods, by means of the reversed passive latex agglutination technique
or EIA technique.33
Helicobacter pylori
For the rapid diagnosis of H. pylori infection, different tech-
niques have been used, since the culture of the biopsy is slow and
demanding.34
Based on H. pylori urease. H. pylori has a potent urease and this
characteristic has been used, since the discovery of H. pylori, for its
diagnosis, in 2 types of assays: rapid urease from the biopsy and
breath assay.
If a piece of biopsy tissue is incorporated into a urea broth or
gel, the colour will change to pink in a few minutes. The urease test
can be used to identify the bacteria obtained by culture, but it is
mainly used as a standard practice in endoscopy units. The first to
be marketed for this purpose was the CLO-test, with which positive
results were obtained between 30 min and 3 h, although it could be
left up to 24 h, which led to a loss of specificity due to other urease
positive bacteria. Currently there are different trademarks, which
seek to shorten the time to obtain the result.35
To perform the urea breath test (UBT), the patient ingests
marked urea and breath samples are collected before and 30 min
after ingestion. If H. pylori is in the stomach, it will hydrolyse
the urea and marked CO2 will be released. A radioactive iso-
tope can be used, 14 C, or a non-radioactive isotope, 13 C, which
is the most widely used and can be measured with a mass or
infrared spectrometer.35 It is the non-invasive test of choice for
the diagnosis and follow-up of patients with H. pylori because of
their high sensitivity and specificity (95–100%) to detect active
infection.35,36
Based on the detection of antibodies. There are numerous sero-
logical tests to detect anti-H. pylori IgG antibodies in the patient’s
serum. The main limitation is that they do not differentiate the
active infection from the past infection and the kit used needs to
be validated locally.35 It is useful for epidemiological studies and
when there is gastrointestinal bleeding, atrophic gastritis, gastric
cancer or MALT lymphoma (mucosa associated lymphoid tissue),
since it can produce a low bacterial load in the stomach and will
reduce the sensitivity of other diagnostic tests.36,37 There are also
tests to detect H. pylori antibodies in urine or saliva, with vari-
able results, so it is currently not recommended for use in patient
management.35,38,39
Based on the detection of antigens in faeces. The stool antigen test
is a non-invasive and qualitative test that detects the H. pylori anti-
gen that has been eliminated in faeces. They can be used: (1) as a
test for the initial diagnosis of infection; (2) for treatment control (at
least 4 weeks after finishing treatment), and (3) for epidemiological
studies.
The first kit to be developed was an EIA with polyclonal
antibodies,39 although it is currently not recommended because
of its lower sensitivity and specificity compared to those that use
monoclonal antibodies.35,39 Of the numerous methods marketed
that use monoclonal antibodies, EIA or ICT present different diag-
nostic precision. Better results are obtained with kits based on EIA
in microwell format39 and are considered equivalent to the UBT in
the Maastricht consensus report.34,35 There is also an automated
chemiluminescent immunoassay with results comparable to the
EIA.34
Sensitivity of 93% and 95% and specificity of 89% and 87%
for the pretreatment diagnosis and sensitivity of 94% and 100%
and specificity of 97% and 91% for post-treatment follow-up were
observed in 2 studies evaluating ICT.35 In a recent study, 3 com-
mercial ICTs were compared and RAPID Hp StAR (Oxoid) was
found to be the most sensitive (91–92%) but less specific (77–85%),
whereas for Uni-GoldTM H.pylori Antigen (Trinity Biotech) and for
ImmunoCard STAT! HpSA (Meridian), a sensitivity of 83% and
a specificity of 90% (87–89%) were observed.40 In other stud-
ies, lower values of sensitivity and/or specificity have also been
observed, therefore it is believed that ICTs need to be improved to be
reliable.40
Based on molecular methods (PCR). For many years, home
molecular methods have been used for the diagnosis of H. pylori
infection from different samples such as gastric biopsy, faeces,
saliva, gastric juice, etc. In addition, they allow for the study
of macrolide resistance mutations such as clarithromycin (23S
rRNA gene), fluoroquinolones (gyrA gene), tetracyclines (16S rRNA
gene), rifabutin (rpoB gene) and amoxicillin (pbp-1a gene),41,42
and have the advantages of speed, greater sensitivity than culture
with phenotypic methods of antibiotic sensitivity, and they detect
heterogeneous resistance, which may be difficult to obtain by
culture.39,43
In the last Maastricht consensus, sensitivity studies on clar-
ithromycin are considered if this antibiotic is to be used as the
first line of treatment in regions with a resistance rate greater than
15%, both by culture and antibiogram, and by a molecular method
directly from the biopsy.36
There are several commercial kits based on real-time PCR
that detect H. pylori directly on the biopsy; other methods, in
addition to H. pylori, detect the major mutations conferring resis-
tance to clarithromycin44–47 (Table 4). There is also a commercial
kit based on PCR followed by reverse line-blot hybridisation
 L. Balsalobre-Arenas, T. Alarcón-Cavero / Enferm Infecc Microbiol Clin. 2017;35(6):367–376 373

Table 4
Commercial systems using molecular methods for the diagnosis of Helicobacter pylori and resistance to clarithromycin or levofloxacin.

Commercial kit Target Technique Samples

Helicobacter pylori genesig Easy Kit Gen rpo H. pylori Real-time PCR Good quality DNA
(genesig) Quantitative
AmpliSens H. pylori-FRT (Ecoli H. pylori End-point PCR Gastric biopsy
s.r.o.) fluorescence detection
Qualitative
VIASURE H. pylori Real Time PCR Gen ureB H. pylori Real-time PCR Gastric biopsy
Detection Kit (CerTest biotec) Qualitative Faeces
Seeplex ClaR-H. pylori ACE Gen 23SrDNA H. pylori PCR-DPO (Dual Priming Gastric biopsy
(Seegene) ClaR mutations Oligonucleotide)
Qualitative
MutaREAL H. pylori kit H. pylori Real-time PCR Gastric biopsy
(Immundiagnostik) ClaR mutations Qualitative
®
RIDA GENE H. pylori (r-biopharm) Gen 16SrDNA H. pylori Real-time PCR Gastric biopsy
ClaR mutations Qualitative
Amplidiag H. pylori + ClariR Gen 23SrDNA H. pylori Real-time PCR Faeces
(Mobidiag) ClaR mutations Qualitative
ClariRes real-time PCR (Ingenetix) Gen 23SrDNA H. pylori Real-time PCR Gastric biopsy
ClaR mutations Qualitative Faeces
GenoType HelicoDR (Hain H. pylori PCR and reverse line-blot Gastric biopsy
Lifescience) ClaR mutations hybridisation
LevR mutations Qualitative

ClaR: resistance to clarithromycin; LevR: resistance to levofloxacin.

(GenoType HelicoDR) which detects resistance to clarithromycin
and fluoroquinolones. Sensitivity of 94% to 100% and specificity
of 86% to 99% have been reported for the detection of clar-
ithromycin resistance and sensitivity of 83% to 87% and specificity
of 95% to 98.5% for fluoroquinolone resistance,39,48 although in
other studies lower levels have been observed for both clar-
ithromycin and fluoroquinolones.39,49 Some of the commercialised
kits can detect H. pylori and its resistance to clarithromycin from
stool samples (Amplidiag H. pylori + ClariR, Mobidiag or ClariRes
real-time PCR, Ingenetix), although no articles have yet beem
published.
significant percentage of mixed infections are detected, more fre-
quent in children under 5 years or in non-hospitalised patients. The
most frequent pathogens in co-infection were Campylobacter and
EPEC.1,53
AllplexTM Gastrointestinal Full Panel Assay (Seegene) is a system
with 4 panels that detects viruses, bacteria and parasites and is
approved for use in Europe (EC marked).
There are more commercial systems such as Nanosphere Veri-
gene (Luminex), Prodesse ProGastro SSCS (Hologic Gen-Probe),
BD MAX enteric bacterial panel (BD), EntericBio real-time Gas-
tro (Serosep), CLART EnteroBac and Enteric Pathogens [EP] Test
(Nanosphere).1,2,50

Syndromic diagnosis of gastroenteritis

Numerous syndromic panels have emerged in recent years
for the microbiological diagnosis of different infectious diseases,
including diarrhoea-producing pathogens. Within a short time
period, they deliver results of all the pathogens included in the
kit and can be a very important tool in some situations, such as
in immunosuppressed patients or serious patients.1 The detection
of most pathogens has a good sensitivity and it is highly valuable
in the detection of coinfections.4
There are different commercial techniques to detect
gastroenteritis-producing pathogens (Table 5), and they vary
widely in the number and type of pathogens they detect and in the
technology they use.1,2,50
Luminex xTAG GPP. It detects bacteria, viruses and para-
sites. FDA approved and with CE marking, 24 samples can be
made in about 5 h in a process that includes: pretreatment,
nucleic acid extraction, multiplex PCR, pellet hybridisation, and
data analysis. This technique increases the rate of positivity
with respect to the conventional methods, and detects mixed
infections.1,51,52
FilmArray GI panel (bioMérieux). It detects bacteria, parasites
and viruses. FDA approved and with EC marking, it consists of
a closed device that requires minimal manipulation. The sys-
tem performs nucleic acid extraction, nested multiplex PCR, and
melting temperature analysis. Performing the test along with
the analysis takes about 60 min. Higher rates of positivity and a
Analysis of volatile organic acids
Volatile Organic Acids are a broad range of stable chemical com-
pounds that are volatile at room temperature and can be detected
in patients’ breath, urine, faeces, and sweat.54 It has proven useful
in the diagnosis of different diseases such as diabetes, gastrointesti-
nal and liver diseases, lung diseases, different types of cancer and in
various infections, such as gastrointestinal infections and H. pylori
infection.55–57
At least 40 volatile acids can be detected in stool samples from
patients with gastroenteritis, regardless of clinical disease, and
different markers are observed depending on the specific type
of pathogen.54 In Bangladeshi patients with Vibrio cholerae infec-
tion, fewer volatile compounds are observed than in healthy donor
samples.58 In a study of 35 patients with infectious diarrhoea,
compared to 6 controls, there were no hydrocarbons and ter-
penes in patients with Campylobacter infection, and the absence
of non-native furan species was indicative of infections with
C. difficile.54
In the exhaled breath analysis of patients infected with H. pylori,
3 volatile compounds (isobutane, 2-butanone, and ethyl acetate)
were observed that were not detected in the controls.54,59 These
compounds can be produced by H. pylori in vivo or be metabo-
lites that the host produces as a result of infection. Elevated levels
of hydrogen cyanide (HCN) and hydrogen nitrate have also been
reported in the exhaled breath of patients with H. pylori gastritis
374 L. Balsalobre-Arenas, T. Alarcón-Cavero / Enferm Infecc Microbiol Clin. 2017;35(6):367–376

Table 5
Pathogens detected in commercial panels for the diagnosis of gastroenteritis.

Virus Bacteria Parasites

FilmArray Rotavirus A Aeromonas Cryptosporidium

(bioMérieux) Norovirus GI/GII Campylobacter Entamoeba
Adenovirus 40/41 Clostridium difficile (toxins A/B) histolytica
Sapovirus Plesiomonas shigelloides Giardia lamblia
Astrovirus Salmonella Cyclospora
Yersinia enterocolitica cayetanensis
Vibrio spp.
Escherichia coli: EAEC, EPEC, ETEC, STEC (stx1 y stx2), E. coli O157,
EIEC/Shigella
xTAG GPP Rotavirus A Campylobacter Cryptosporidium
(Luminex) Norovirus GI/GII C. difficile (toxins A/B) E. histolytica
Adenovirus 40/41 Salmonella G. lamblia
Y. enterocolitica
Vibrio spp.
E. coli: ETEC, STEC (stx1 y stx2), E. coli 0157, EIEC/Shigella
Verigene Rotavirus A Campylobacter
(Luminex) Norovirus GI/GII Salmonella
Y. enterocolitica
Vibrio spp.
E. coli: STEC (stx1 y stx2), ECEI/Shigella
Seeplex Panel 1 Panel 2 Panel 4
(Seegene) Norovirus GI Salmonella spp. G. lamblia
Norovirus GII EIEC/Shigella spp. E. histolytica
Rotavirus Vibrio spp. Cryptosporidium
Adenovirus Campylobacter spp. spp.
Astrovirus C. difficile Toxin B Blastocystis hominis
Sapovirus Y. enterocolitica Dientamoeba
Aeromonas spp. fragilis
Panel 3 C. cayetanensis
Hypervirulent C. difficile
E. coli: E. coli O157:H7, EHEC (stx1/2), EPEC (eaeA), ETEC (It/st), EAEC
(aggR)
®
RIDA GENE Norovirus GI/GII Campylobacter spp. G. lamblia
(R-Biopharm) Rotavirus Salmonella spp. Cryptosporidium
Y. enterocolitica parvum, E.
C. difficile (TcdA, TcdB) histolytica
E. coli: EHEC, EPEC, ETEC, EIEC/Shigella spp. D. fragilis
Faecal pathogens A Rotavirus A Salmonella spp. G. lamblia
(AusDiagnostics) Norovirus G1/G2, Shigella spp. Cryptosporidium
Adenovirus F/G Campylobacter spp. D. fragilis, E.
C. difficile (toxin B) histolytica
Y. enterocolitica B. hominis
Aeromonas hydrophila
Enteric Pathogens Norovirus, Rotavirus Campylobacter spp.
[EP] Test Salmonella spp.
(Nanosphere) Shigella spp.
STEC (stx1/stx2)
Vibrio spp.
Y. enterocolitica

compared to healthy controls. However, HCN is not specific to H. pathogens simultaneously can be very useful in certain groups of
pylori, as it can be detected in exhaled breath samples from patients patients and will become increasingly important because of their
infected with Pseudomonas aeruginosa.54 high sensitivity and specificity, as well as their speed, albeit at a
higher cost.

Conclusions Conflicts of interest

Rapid techniques are extremely valuable in the diagnosis of gas-
trointestinal infections. Those based on the detection of antigens
are already common practice in clinical microbiology laborato-
ries, as are those based on molecular techniques, although only
for some pathogens such as C. difficile and Shiga toxin-producing
E. coli. When choosing the technique and trademark to use, we
must consider different criteria, which will include: the pathogen
or pathogens we want to detect, the type of patients for whom it
is intended, the number of requests we think need to be made, the
availability of equipment in the department, as well as the cost of
the technique. If we are going to conduct a small number of tests,
individual techniques such as EIA or ICT would be most appropri-
ate, while if the number of tests is high, it will be more convenient
to perform ELISA tests. Molecular techniques that detect different
T.A.C. has received sporadic support for attendance at con-
gresses of Hain Lifesciencie, bioMerieux, Meridian.
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